Nanotechnology of materials, related microbiological means of manipulation, and synthetic biology require a means to directly observe nanoscale interactions and classify nano-material morphological properties in solution on a small scale. More specifically the lack of particle and colloid size descriptors and in situ nanoscale imaging techniques are detrimental to advancing the state of the art because it inhibits the ability to verify the reproducibility of nano-fluid preparation necessary for research groups to compare properties.
Using and relying only on more conventional imaging techniques such as a transmission electron microscope (TEM) and a scanning electron microscope (SEM) raises several concerns. TEM and SEM samples are dried and exposed to vacuum before being imaged which changes the observed fundamental properties of the sample. Further, when using a wet cell with a TEM, images should be taken immediately since the energy of the beam quickly de-hydrates the small amount of fluid within the sample. Using conventional techniques, this does not lend itself to accurate particle and agglomerate size characterization much less quantification of other physio-chemical properties.
Besides standard TEM use, the current suite of options available to observe objects at the nanoscale in situ includes the electron cryo-microscope, dynamic light scattering (DLS), small angle x-ray scattering (SAXS), small angle neutron scattering (SANS), and others adapted for more specific applications. The electron cryo-microscope presents drawbacks to drying similar to the TEM and SEM as the sample must be flash frozen into vitreous ice. The procedure distorts the image as well as limiting sample thickness that may be addressed. Moreover, it is undesirable to freeze a sample because the properties of the solution, including species interaction and separation, change. Dynamic light scattering is a very reliable way to determine particle size. DLS works with a broad range of materials in most instances. However, DLS does not work well with non-spherical species such as carbon nanotubes or DNA. Like DLS, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS) are broadly used in research and industry. SAXS has a rather low observable size limit, not able to detect the length of most carbon nanotubes (CNTs), and is best suited for analyzing the surface and bulk properties of larger samples. SANS shares some of the undesirable traits of SAXS and adds the threat of sample damage via neutron-particle interaction. This degrades observation of the extent of colloidal agglomeration, for example.
Existing methods, devices, developments and publications are inherently limited. U.S. Pat. No. 4,071,766, to Kalman et al., employs a film-sealed micro-chamber to hold a wet sample. There are bores or pipes to introduce fluid to this micro-chamber. However, the large chamber or gap height (the length that the electron beam needs to traverse) makes it impractical for use in transmission electron microscopy. For medium high-voltage TEMs, typical sample thickness which is transparent to electron beam is below 700 nm.
Fukami et al. use a design similar to Kalman et al. but with an assembly comprising multiple parts to form the chamber instead of one integral part. A. Fukami and K. Fukushima, Proc. Eighth European Congress on Electron Microscopy, Budapest, pp. 71-80, 1984. A Fukami, K. Fukushima and N. Kohyama, Observation Techniques for Wet Clay Minerals Using Film-Sealed Environmental Cell Equipment Attached to High-Resolution Electron Microscope, In: Microstructure of Fine-Grained Sediments from Mud to Scale, R. H. Bennett, W. R. Bryant, M. H. Hulbert (eds.), New York: Springer-Verlag, pp. 321-331, 1991. Carbon films supported by copper grids are used as the sealing film. Again, the spacing between the films is large. The film is easily broken which can result in extreme damage to microscopy equipment.
U.S. Pat. No. 5,406,087, to Fujiyoshi et al., describes a specimen-holding device that is quite similar to the Fukami et al. design. Two polymer films are pressed together directly by backing copper grids and the samples are trapped between the films. The polymer films and the thin copper grids tend to deform making it impossible to form thin liquid films over relatively large areas. Further, the film is easily broken which can result in extreme damage to microscopy equipment. Moreover, the design does not have any element to control the size of the gap and requires accurate alignment of the grids, which is difficult.
Williamson et al. construct a wet cell by gluing two Si3N4-coated silicon wafers face to face. M. J. Williamson, R. M. Tromp, P. M. Vereecken, R. Hull, and F. M. Ross, Dynamic Microsocopy of Nanoscale Cluster Growth at the Solid-Liquid Interface, Nature Materials, Vol. 21, pp. 532-536, August 2003. Each wafer has a Si3N4 thin film membrane window formed by selective etching. The wafers are adhered to one another around the edges and a gap between the wafers is created with a deposited SiO2 spacer element. Liquid is loaded into the cell through ports on one wafer. The cell is sealed by gluing sapphire plates over the ports. This invention involves a tedious and time-consuming process involving multiple gluing and curing steps. Further, the glue tends to enlarge the gap. This device does not provide for a sample that is thin enough for observation of desired characteristics.
The following U.S. patents suffer from similar drawbacks as discussed above: U.S. Pat. No. 7,476,871 to Chao et al.; U.S. Pat. No. 7,304,313 to Moses et al.; U.S. Pat. No. 7,253,418 to Moses et al.; U.S. Pat. No. 7,230,242 to Behar et al.; U.S. Pat. No. 7,219,565 to Fischione et al.; U.S. Pat. No. 6,992,300 to Moses et al.; U.S. Pat. No. 6,989,542 to Moses et al.; U.S. Pat. No. 5,412,211 to Knowles; U.S. Pat. No. 5,362,964 to Knowles et al.; U.S. Pat. No. 5,097,134 to Kimoto et al.; and U.S. Pat. No. 4,705,949 to Grimes et al.
A process capable of allowing one to analyze in situ samples of a broad nature mitigates many of the above limitations of conventional methods. Further, it would be advantageous to have a sample holding device with the following characteristics: self-aligning windows on holder pieces and wafers, thus providing for ease of assembling the sample in the holder; a controllable sample volume (gap) thickness via modifying spacer (integral to the wafer or separate) thickness; uniform sample volume thickness; improved spatial resolution resulting from reduced gap (sample volume) thickness; improved resolution resulting from use of ultra-thin (<100 nm) membranes as windows, in particular Si3N4-coated membranes; durable and reliable sealing of the sample combined with a window pattern that provides reinforcement resulting in safe use in vacuum environments.
A reliable readily implemented in situ imaging technique embodied in select embodiments of the present invention has the above characteristics and allows direct examination of nano-fluid samples without affecting basic characteristics, such as nano-particle dispersion.